Abstract
Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. Palmitoylation is mediated by a family of at least 23 palmitoyl acyltransferases (PATs) characterized by an Asp-His-His-Cys (DHHC) motif. Many palmitoylated proteins have been identified, but PAT-substrate relationships are mostly unknown. Here we present a method called palmitoyl-cysteine isolation capture and analysis (or PICA) to identify PAT-substrate relationships in a living vertebrate system and demonstrate its effectiveness by identifying CKAP4/p63 as a substrate of DHHC2, a putative tumor suppressor.
Highlights
Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage
Reactive thiols are protected by modification with methyl methanethiosulfonate (MMTS), palmitoyl-cysteine thioester bonds were selectively cleaved by neutral hydroxylamine (NH2OH), yielding a distinct set of free, reactive thiols comprised of formerly palmitoylated cysteines
Using Palmitoyl-cysteine isolation capture and analysis (PICA) we identified CKAP4/p63 as a physiologically important substrate of DHHC2, the palmitoyl acyltransferases (PATs) encoded by ZDHHC2
Summary
Protein palmitoylation is the post-translational addition of the 16-carbon fatty acid palmitate to specific cysteine residues by a labile thioester linkage. The ability of PICA to identify PAT substrates is based on the principle that it quantifies the differential frequency of palmitoylation of individual proteins in control conditions versus conditions in which the function of a single PAT is reduced by siRNA-mediated gene knockdown (Fig. 1).
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